Abstract
Autophagy is typically activated in cancer cells as a rescue strategy in response to cellular stress (e.g., chemotherapy). Herein, we found that Berbamine Hydrochloride (Ber) can act as an effective inhibitor of the late stage of autophagic flux, thereby potentiating the killing effect of chemotherapy agents. Lung carcinoma cells exposed to Ber exhibited increased autophagosomes, marked by LC3-II upregulation. The increased level of p62 after Ber treatment indicated that the autophagic flux was blocked at the late stage. The lysosome staining assay and cathepsin maturation detection indicated impaired lysosomal acidification. We found that Nox2 exhibited intensified co-localization with lysosomes in Ber-treated cells. Nox2 is a key enzyme for superoxide anion production capable of transferring electrons into the lysosomal lumen, thereby neutralizing the inner protons; this might explain the aberrant acidification. This hypothesis is further supported by the observed reversal of lysosomal cathepsin maturation by Nox2 inhibitors. Finally, Ber combined with cisplatin exhibited a synergistic killing effect on lung carcinoma cells. Further data suggested that lung carcinoma cells co-treated with Ber and cisplatin accumulated excessive reactive oxygen species (ROS), which typically activated MAPK-mediated mitochondria-dependent apoptosis. The enhanced anti-cancer effect of Ber combined with cisplatin was also confirmed in an in vivo xenograft mouse model. These findings indicate that Ber might be a promising adjuvant for enhancing the cancer cell killing effect of chemotherapy via the inhibition of autophagy. In this process, Nox2 might be a significant mediator of Ber-induced aberrant lysosomal acidification.
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The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Abbreviations
- Ber:
-
Berbamine Hydrochloride;
- Cis:
-
Cisplatin
- PTX:
-
Paclitaxel
- ATCC:
-
American Type Culture Collection
- DMEM:
-
Dulbecco’s Modified Eagle Medium
- CCK8:
-
Cell Counting Kit-8
- Baf:
-
Bafilomycin A1; CQ, chloroquine
- Rapa:
-
Rapamycin
- HBSS:
-
Hank’s Balanced Salt Solution
- AO:
-
Acridine orange
- Cat L:
-
Cathepsin L
- Cat D:
-
Cathepsin D
- V-ATPase:
-
Vacuolar H + -ATPase
- ROS:
-
Reactive oxygen species
- Ras-related C3 botulinum toxin substrate 2:
-
Rac2
- NADPH oxidase:
-
Nox
- NAC:
-
N-Acetylcysteine
- PARP:
-
Poly (ADP-ribose) polymerase
- PI:
-
Propidium iodide
- cyto-c:
-
Cytochrome c
- mito:
-
Mitochondria
- cyto:
-
Cytosol
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Funding
This work was supported by the National Natural Science Foundation of China (Grant No. 82074070, 82104571), the Guangdong Natural Science Foundation (Grant No. 2020A1515110632), the Science and Technology Plan Project of Guangzhou of China (Grant No. 202102020721), the Guangdong Provincial Bureau of Traditional Chinese Medicine Research Foundation (Grant No. 20222043, 20221119), 2019 characteristic innovation projects of Guangdong colleges and Universities (Grant No. 2019GKTSCX045), the Guangdong Medical Science and Technology Research Foundation (Grant No. A2020293), and the Bethune Quest-Pharmaceutical Research Capacity Building Project (Grant No. B-19-H-20200622).
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KW and JYX were involved in conceptualization. YJZ, QGC, and YS helped in methodology. YJZ and QGC contributed to validation and formal analysis. XLW was involved in resources and funding acquisition. TXZ and WBZ helped in project administration and data curation. BNC and YS contributed to visualization and software. CXL and YJJ was involved in investigation. YHT and BYD helped in supervision. YJZ was involved in writing—original draft preparation. JYX and KW helped in writing—review and editing and funding acquisition.
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The animal experiment was approved by the Institutional Animal Care and Use Committee at Guangzhou University of Chinese Medicine (Project No. 20190927002).
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Headlights
• Berbamine Hydrochloride (Ber) accelerated the assembly of the Nox2 complex by promoting the activity of Rac2, thereby impairing lysosomal acidification in lung cancer cells.
• Ber potentiated the killing effect of cisplatin through the ROS-MAPK signaling pathway in lung cancer cells.
• The enhancive anti-cancer effect of Ber combined with cisplatin was also confirmed in an in vivo xenograft mouse model.
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Zhan, Y., Chen, Q., Song, Y. et al. Berbamine Hydrochloride inhibits lysosomal acidification by activating Nox2 to potentiate chemotherapy-induced apoptosis via the ROS-MAPK pathway in human lung carcinoma cells. Cell Biol Toxicol 39, 1297–1317 (2023). https://doi.org/10.1007/s10565-022-09756-8
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DOI: https://doi.org/10.1007/s10565-022-09756-8